Molecular cloning: 6 RBS+ arac/ laci

Transcription

1 Molecular cloning: 6 RBS+ arac/ laci Resource: 6 RBS: from the parts: B0030, B0032, B0034, J61100, J61107, J61127; renamed as RBS1 RBS2 RBS6; arac: C0080 laci: C0012 July 6 th Plasmid mini prep: 6 RBS; laci; arac; Double digest: 6 RBS: Spe1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL laci, arac: Xba1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL 37 4 hour July 7 th Products of double digest of laci and arac, marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb voltage and time: 60V 5min; 120V 15min lane1: laci plasmid; lane2: arac plasmid; lane3: marker; lane4: digested product of laci; lane5: digested product of arac; 1 / 12

2 DNA Gel purification: laci and arac PCR product purification: 6 RBS DNA ligation: System 10uL: Insert 4uL, vector 1uL, water 3uL, buffer 1uL, T4 DNA ligase 1uL 16 4 hour Insert: laci; arac; tetr (from Min Lin); Vertor: 6 RBS Transformation: Products of ligation, competent cells 50uL each, Smear to LB plate with Amp July 8 th Every plate is very well: more than 100 clones Waiting for PCR to check the positive clones July 9 th PCR: Master mix 5ul, primer (standard primer) 0.5uL each, template; July 10 th 2 / 12

3 Products of PCR marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb voltage and time: 60V 5min; 120V 15min Up row: lane 1~3: tetr+rbs1 1~3; lane 4~6: tetr+rbs2 1~3; lane 7~9: tetr+rbs3 1~3; lane 10: marker; lane 11~12: tetr+rbs4 2~3; lane 13~15: tetr+rbs5 1~3; lane 16~18: tetr+rbs6 1~3; down row: lane 1~3: laci+rbs1 1~3; lane 4~6: laci+rbs2 1~3; lane 7~9: laci+rbs3 1~3; lane 10: marker; lane 11~12: laci+rbs4 2~3; lane 13~15: laci+rbs5 1~3; lane 16~18: laci+rbs6 1~3; result: 10 clones were successfully constructed: RBS1~5+lacI and tetr; T hey a re pa r t s : K , K , K , K , K , 3 / 12

4 2 clones were failed: RBS6+lacI and tetr. Molecular cloning: RBS-lacI/tetR + terminator Resource: RBS-LacI: myself: 2 B0034-C0012, renamed as L1, L2 RBS-tetR: myself: 2 B0034-C0040, renamed as t2, t3 Terminator: Haoqian Zhang & Guosheng Zhang: B0015 July 10 th Plasmid mini prep: 6 RBS-tetR: RBS1-tetR1; RBS2-tetR3; RBS3-tetR2, 3 (t2, t3); RBS4-tetR2; RBS5-tetR2; 6 RBS-lacI: RBS1-lacI1; RBS2-lacI1; RBS3-lacI1, 2 (L1, L2); RBS4-lacI3; RBS5-lacI2; Double digest: L1, L2, t2, t3: Spe1 1uL, EcoR1 1uL, plasmid 4uL, Buffer 2uL, water 12uL 37 4 hour Products of double digest of L1, L2, t2, t3, Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6 Voltage and time: 60V 5min; 120V 15min Lane1: t2: insert 700bp; Lane2: t3: insert 700bp; Lane3: marker; Lane5: L1: insert 1.1kb; 4 / 12

5 Lane6: L2: insert 1.1kb; DNA Gel purification: I made a big mistake here. I purified the brightest ones, which are vectors. So I do the double digest again. Double digest (again): L1, L2, t2, t3: Spe1 1uL, EcoR1 1uL, plasmid 4uL, Buffer 2uL, water 12uL 37 over night. July 11 th Gel electrophoresis (again): Products of double digest of L1, L2, t2, t3, Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6 Voltage and time: 60V 5min; 120V 15min Lane1: L1: insert 1.1kb; Lane2: L2: insert 1.1kb; Lane3: marker; Lane5: t2: insert 700bp; Lane6: t3: insert 700bp; 5 / 12

6 DNA ligation: System 10uL: Insert 6uL, vector 2uL, buffer 1uL, T4 DNA ligase 1uL 16 4 hour Insert: L1 L2 t2 t3; Vector: terminator digested by EcoR1 and Xba1 (provided by Haoqian Zhang & Guosheng Zhang) Transformation: (by Min Lin) Products of ligation, competent cells 50uL each, Smear to LB plate with Amp July 12 th Every plate is very well: more than 100 clones PCR: 14 tubes: L1 3+L2 3+t2 3+t3 3 and 2 negative controls Master mix 5ul each, primer (standard primer) 0.5uL each, template; Products of PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Voltage and time: 60V 5min; 120V 15min 6 / 12

7 Lane 1: t2-1; Lane 2~4: t3-3~1; Lane 5: t2-3; Lane 7: t2-1+2; Lane 8: negative control1; Lane 9: marker; Lane 10: negative control2; Lane 11~13: L2-3~1; Lane 16~18: L1-3~1; Result: There is a polluted line at about 1kb place, but it did not confuse us. The right place of L1 & L2 is about 1.3kb and of t2 & t3 is about 800bp. L1-1~3, L2-1~3, t2-1&3 and t3-1~2 should be the positive clones. 4 clones were successfully constructed: L1 & L2: 2 B0034-C0012-B0015 T2 & t3: 2 B0034-C0040-B0015 By Shuke Wu Molecular cloning: 2M-lacI/tetR-term+Pcat Parts: K228813/14+I14033=K228815/16 Resource: 2m-lacI/tetR-term: myself, rename as L1, L2, T1, T2 Pcat: part I14033 July13 th Plasmid mini prep: I14033: Pcat; 7 / 12

8 K228813: L1, L2; K22814: T1 T2; Double digest: Pcat: Spe1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL L1, L2 T1, T2: Xba1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL 37 4 hour Products of double digest of L1, L2, T1, T2, marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb voltage and time: 60V 5min; 120V 15min lane1: digested product of L1; lane2: digested product of L2; lane3: marker; lane4: digested product of T1; lane5: digested product of T2; I found that they are not digested well. There are not any insert. There is possibility that I added the wrong enzymes or something wrong in the enzymes. Repeat digest. Double digest: (again) Pcat: Spe1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL L1, L2 T1, T2: Xba1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL 37 4 hour (again) Products of double digest of L1, L2, T1, T2, marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb 8 / 12

9 Voltage and time: 60V 5min; 120V 15min lane1: digested product of L1; lane2: digested product of L2; lane3: marker; lane4: digested product of T1; lane5: digested product of T2; I can found the inserts of L1, L2, and they about 1.3kb. I can found the inserts of T1, T2, and they about 800bp. DNA Gel purification: Inserts of L1, L2, T1, T2. PCR product purification: Product of digest: Pcat. DNA ligation: System 10uL: Insert 6uL, vector 2uL, water 3uL, buffer 1uL, T4 DNA ligase 1uL 16 overnight. Insert: L1, L2, T1, T2; Vertor: Pcat July 14 th Transformation: Products of ligation, competent cells 50uL each, Smear to LB plate with Kan The colonies are too small at night, and let they grow overnight. July 15 th Every plate is very well: more than 100 clones PCR: (colony PCR) 9 / 12

10 System 10 ul: Master mix 5ul, primer (standard primer) 0.5uL each, water: 4uL template; Products of PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Voltage and time: 60V 5min; 120V 15min lane 1~3: L1 1~3; lane 4~6: L2 1~3; lane 7: PCR control. lane 8: marker; lane 9~11: T1 1~3; lane 12~14: T2 1~3; Right colonies: L1 L2: 1.4kb; T1, T2 900bp result: 2 clones were successfully constructed: 2M-tetR-term+Pcat *2 They are parts: K Unexpected result: L1, L2 failed, due to reversing PCR from July 16 th to July 20 th. Because use the reversing primers can not amplify the plasmid of L1 L2, the clone here should be K228813: 2M-lacI-term, not K228815: Pcat-2M-lacI-term. For more detail, please refer to my experiment notes reversing July 16 th ~July 21 st. Repeat clone 2M-lacI-term+Pcat: July 17 th Double digest: Pcat: Spe1 1uL, Pst1 1uL, plasmid 4uL, Buffer 2uL, water 12uL PCR product purification: Product of digest: Pcat. 10 / 12

11 DNA ligation: System 10uL: Insert 6uL, vector 2uL, water 3uL, buffer 1uL, T4 DNA ligase 1uL 16 overnight. Insert: L1, L2,(use the inserts on the July 13 th ) Vector: Pcat. July 18 th Transformation: Products of ligation, competent cells 50uL each, Smear to LB plate with Kan July 19 th Every plate is very well: more than 100 clones PCR: (colony PCR) System 10 ul: Master mix 5ul, primer (reversing primers) 0.5uL each, water: 4uL; template; Because reversing primers can bind to Pcat and terminator, they can be use to check the right colonies. For more details, refer to my experiment notes reversing July 16 th ~July 21 st. Products of PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Voltage and time: 60V 5min; 120V 45min Lane1~8: colonies 1~8; Lane9, 10: other plasmid controls; 11 / 12

12 Lane11, 12: no template controls. Result: We can found the 3 rd colony is right. So I successfully constructed: 2M-tetR-term+Pcat, it is the part K BY Shuke Wu 12 / 12